Pulmonary endothelium is a critical, semi-selective cellular barrier, which prevents the leakage of water, solutes, macromolecules and cells from the vasculature. Compromised pulmonary endothelial cell (EC) barrier integrity caused by mechanical ventilation at high tidal volumes is associated with altered patterns of cyclic stretch experienced by EC, and leads to increased permeability, alveolar flooding, leukocyte infiltration, hypoxemia, and increased morbidity and mortality. We have previously identified cytoskeletal mechanisms of EC permeability changes caused by barrier-protective (sphingosine 1-phosphate) and barrier-disruptive (thrombin) agents and described a critical involvement of small GTPases Rac and Rho in remodeling of cell focal adhesions and adherens junctions associated with EC barrier regulation. Our published results and preliminary data strongly suggest, that modulation of Rac and Rho activities by mechanical stimuli significantly impacts agonist-induced regulation of EC permeability. Our original hypothesis is that magnitude-dependent regulation of Rac and Rho GTPases by cyclic stretch (CS) may determine severity of the pulmonary EC barrier dysfunction and rates of barrier restoration during mechanical ventilation at high tidal volume. We also hypothesize that interactions of cytoskeletal (actin, cortactin), focal adhesion (paxillin, PKL/GIT2), and adherens junction (VE-cadherin, beta-catenin) effectors of small GTPases may be critically involved in the lung barrier regulation by mechanical forces. Specific Aim #1 will determine physiological and pathologically relevant levels of CS experienced by pulmonary EC using combination of in vitro and in vivo approaches and will explore a link between magnitude-dependent Rac and Rho regulation and EC cytoskeletal changes. Specific Aim #2 will investigate a role of physiological and pathological CS levels in the agonist-induced regulation of EC cytoskeleton, cell contacts, and monolayer integrity. Specific Aim #3 will identify molecular mechanisms of mechanochemical regulation of small GTPase activities by Rac- and Rho-specific GEFs (Tiam1, Vav2, betaPIX, GEF-H1 and p115-RhoGEF). We speculate that the results obtained in this study will significantly impact our understanding of the role of small GTPases in the EC adaptation to alterations of mechanochemical environment via specific mechanisms involving cytoskeletal and cell adhesion remodeling (SA#1 and SA#2), discover novel mechanisms of GEF-mediated regulation of Rac and Rho by mechanochemical stimuli (SA#3), and allow us to develop new insights into cellular mechanisms underlying endothelial dysfunction in ventilator-induced lung injury.